Spatial distribution of three ARGONAUTEs regulates the anther phasiRNA pathway

Argonaute protein (AGO) in association with small RNAs is the core machinery of RNA silencing, an essential mechanism for precise development and defense against pathogens in many organisms. Here, we identified two AGOs in rice anthers, AGO1b and AGO1d, that interact with phased small interfering RNAs (phasiRNAs) derived from numerous long non-coding RNAs. Moreover, 3D-immunoimaging and mutant analysis indicated that rice AGO1b and AGO1d cell type-specifically regulate anther development by acting as mobile carriers of these phasiRNAs from the somatic cell layers to the germ cells in anthers. Our study also highlights a new mode of reproductive RNA silencing via the specific nuclear and cytoplasmic localization of three AGOs, AGO1b, AGO1d, and MEL1, in rice pollen mother cells.

Argonaute proteins (AGOs) are essential for the epigenetic regulation of plant development. AGO1 generally binds to miRNA to function in post-transcriptional gene silencing. The authors identified the function of two AGOs" AGO1b, and AGO1d, which interact with phased small interfering RNAs (phasiRNAs) to regulate rice anther development by acting as mobile carriers of the phasiRNAs from the somatic cell layers to the germ cells in anthers. They also performed three-dimensional (3D)-immunostaining using whole anthers, which to identify the specific subcellular localization of AGO1b/d in each somatic cell layer and germ cell. The authors emphasized a reproductive RNA silencing mechanism via the specific nuclear and cytoplasmic localization of AGO1b, AGO1d, and MEL1, in the anther phasiRNA pathway of rice pollen mother cells. However, the experiments could not support their conclusions, and more solid data are needed.
Major comments: 1. The author only performed the 3D multiple immunostaining to show the differing intracellular localization of AGO1b in the nucleus and AGO1d in the cytoplasm of all four cell types in the anther wall. However, The evidence provided in the paper is insufficient and needs to be confirmed by more methods. Such as confirmed by the immune colloidal gold technique. 2. The author showed that AGO1b mRNA was expressed in the outer layers of the anther wall, and not in the inner tapetum layers or pollen mother cells. And AGO1d mRNA was also absent in the pollen mother cells by the immunostaining and RNA in situ hybridization assays, they are not enough to prove the conclusion. The quality of Fig.4 and Fig.5a-f is not good, and the RNA in situ hybridization signals cannot be clearly distinguished. Since it cannot be proved that AGO1b and AGO1d are only expressed in somatic cells, it cannot be said that they can be transferred to the mother cells. On the other way, from the result of Tokunori Hobo et al., 2008and Tang et al., 2010. (Tokunori Hobo et al., 2008, Various spatiotemporal expression profiles of anther-expressed genes in rice; Tang et al., 2010, Global gene profiling of laser-captured pollen mother cells indicates molecular pathways and gene subfamilies involved in rice meiosis. Plant Physiology), AGO1b, and AGO1d mRNA was expressed in the pollen mother cells. Therefore, more evidence is needed to confirm this result. Therefore, it could not be confirmed that the two genes translated into proteins and moved to the pollen mother cells. 3. The different localization of mRNA and protein of AGO1b/d subfamily genes in anther suggests that AGO1b and AGO1d migrate from outer layers to inner layers and pollen mother cells as carriers of phasiRNA. The authors did not separate the tissues from each layer of pollen mother cells for RNA -immunoprecipitation. It is not solid enough that the experimental evidence to support this conclusion. 4. The experimental technology applied in the full text is relatively limited, actually, it does not clarify the significance and function of these two proteins to the AGO family or for the anther development in rice. Minor comments: 1. Figure 1d, The phenotypes of the six anthers of ago1b-1 ago1d-3 are not completely consistent, what is the reason# 2. In fact, the author did not do any experiments to prove that ARGONAUTE-mediated RNA silencing in anther development" so the title of this manuscript is not appropriate.

Responses to reviewers' comments
First of all, we would like to thank the reviewers for their constructive and valuable comments with regard to our manuscript. We have indicated the major changes made in the revised manuscript, using yellow highlighting, and provided point-by-point responses to the reviewers' comments below.
Additional results and major changes 1. 3D anther imaging to differenciate the internal structure in mutant and wild type anthers ( Fig. 1h  9. qPCR analysis of AGO1b, AGO1d, and MEL1 expression in pollen mother cells (Fig. 6d). The numbers of all seeds and fertile seeds were counted in each panicle, and we calculated the fertility rate per panicle as fertile seed numbers/total seed numbers. More than three panicles were counted for one biological replicate. Finally, fertility data shown in box plots are from more than three biological replicates for each mutant/control line (Nipponbare, WT, ago1b-1, ago1b-2, ago1b-3, ago1d-1, ago1d-2, ago1d-3, and ago1b-1 ago1d-3) for Fig. 1c and Supplementary Fig. 2a

YNU]T&
We agree and have removed the statement about SDS-PAGE.
Anther  5) to analyze the localization of AGO1b, AGO1d, and MEL1 at the same time with high resolution. We performed high-resolution multiple immunoimaging with these three AGOs' antibodies and captured images using the Airyscan function of LSM 880 (Fig. 5), which enabled us to analyze samples with a resolution around half of the regular confocal system (i.e., 120 nm). These imaging results permitted spatial discrimination, including cellular and subcellular localization, between three AGOs, AGO1b, AGO1d, and MEL1 ( Fig. 5a-h; page 9, line 14 -page 10, line 11).
We have also added negative-control imaging in this revision, in which samples were not treated with primary antibodies for AGO1b/d or MEL1, but were treated with secondary antibodies ( Supplementary Fig. 6). Detection only of DAPI signals in the negative controls confirms that fluorescence from the immunostaining reflects the localization of the respective AGOs, and not autofluorescence (page 10, line 1-6).
Thus, this high-resolution immunoimaging with negative controls (Fig. 5 and Supplementary   Fig. 6) provides evidence for the specific cellular and subcellular localization of three AGOs in addition to the conclusions drawn from 3D immunoimaging (Fig. 4). We have added the imaging data and described them in the revision ( Fig. 5 We thank the reviewer for this comment and the microarray information. In response, we have added in situ hybridization data for MEL1 mRNA localization as a PMC-specific marker (Fig. 6c), and we also captured clear images for AGO1b, AGO1d, and MEL1 by adjusting the microscope to the most appropriate setting. We have also added in situ hybridization images of four anther locules and negative controls, to ensure the quality of the in situ hybridization (Supplementally Fig. 7). The In addition to the new in situ hybridization data, we performed qPCR using RNAs extracted from pollen mother cells in this revision. MEL1 was highly expressed but we found extremely low expression of AGO1b and AGO1d ( Fig. 6d;  Xiaofeng Cao's group). We also published this manuscript as a preprint before the Cao group's work We agree with the reviewer, and for the revised manuscript, we performed high-throughput RNA sequencing using total RNAs extracted from 0.5-mm anthers of WT and ago1b-1 ago1d-3 mutants 5JJOZOUTGRR_$ we further analyzed the anthers using 3D histological imaging methods for entire rice anthers, which can be used for distinguishing the internal structure of the anthers at the post-meiotic stage ( Fig. 1h-o). Even though the ago1b/d double mutant has an outwardly normal anther structure, it showed abnormalities in the internal structures of the anther. The pollen grains were not filled in parts of the anther locule, some grains were crushed, and development of the inner layer was partially abnormal in 0.9-mm anthers ( Fig. 1h-o). We have changed the title to "Spatial distribution of three ARGONAUTEs regulates the anther phasiRNA pathway".
Reviewer #1 (Remarks to the Author): The authors have successfully addressed all of my concerns.
Reviewer #2 (Remarks to the Author): Thanks to the authors for providing the additional experiments. Since this study highlights a new mode of reproductive RNA silencing via the specific nuclear and cytoplasmic localization of three AGOs, AGO1b, AGO1d, and MEL1, in rice pollen mother cells. More solid evidence is needed. Unfortunately, the present evidence even for mobile localization is slim.
Major comments# 1"Though the author generated a guinea pig MEL1 antibody( Supplementary Fig. 5) to analyze the localization of AGO1b, AGO1d, and MEL1 at the same time with high resolution. The redone supplementary experiment cannot directly explain the mobile function of AGO1b and AGO1d. The analysis of AGO1b, AGO1d, and MEL1 localization by immune gold electron microscopy using anti-AGO1b, AGO1d, MEL1 antibodies in developing anthers can explain the mobile function of AGO1b and AGO1d more directly. It is suggested to supply this experiment. 2"In situ hybridization experiment still needs to be improved, and the specific pollen stage and development stage can not be seen from the cell structure. First of all, we would like to thank the reviewers for their constructive and valuable comments with regard to our manuscript. We have indicated (highlighted in yellow) the major changes made in the second revised manuscript and responded to Reviewer #2's comments below.
Additional results and major changes 1) New images for in situ hybridization, using a new probe to distinguish the four cell layers of anther walls ( Fig. 6b; Supplementary Fig. 7c and d; Supplementary Table 10).
Reviewer #1 (Remarks to the Author): The authors have successfully addressed all of my concerns.
Reviewer #2 (Remarks to the Author): Thanks to the authors for providing the additional experiments. Since this study highlights a new mode of reproductive RNA silencing via the specific nuclear and cytoplasmic localization of three AGOs, AGO1b, AGO1d, and MEL1, in rice pollen mother cells. More solid evidence is needed. Unfortunately, the present evidence even for mobile localization is slim.
Major comments# 1"Though the author generated a guinea pig MEL1 antibody( Supplementary Fig. 5) to analyze the localization of AGO1b, AGO1d, and MEL1 at the same time with high resolution. The redone supplementary experiment cannot directly explain the mobile function of AGO1b and AGO1d. The analysis of AGO1b, AGO1d, and MEL1 localization by immune gold electron microscopy using anti-AGO1b, AGO1d, MEL1 antibodies in developing anthers can explain the mobile function of AGO1b and AGO1d more directly. It is suggested to supply this experiment.
We thank Reviewer#2 for this suggestion. In this study, we indicated the cell-type difference of mRNA and protein localization between somatic anther wall (SOMA) and pollen mother cells (GERM) for the mobile AGO1b/d mechanism. Cell-and tissue-level dynamic change is a key factor in mobile studies, rather than organelle-level (i.e., subcellular) resolution. We would like to emphasize that 'mobile AGO1b/d' here refers to the movement of these proteins between cells/tissues, and not to their movement between compartments within the same cell. Reviewer#2 suggests the use of immunogold electron microscopy at a high-resolution level instead, but we don't understand -and the reviewer doesn't explain -what could be observed in this system that would elucidate such an inter-cell mobile function. In the first revision, we have already added negative control data and new high-resolution data to reveal the cell-level differential localization of AGO1b/d as mobile proteins. Thus, we think there is little to be gained by using a higher-resolution level for illuminating mobile functions. To clarify mobile molecular functions, the behavior of regulators plays an essential role (Taoka et al., 2011 Nature;Kitagawa et al., 2022 Science). We are interested in understanding the mobile molecular functions of AGO1b/d and are now initiating studies that focus on candidate AGO1b/d regulators. We hope our subsequent research will contribute to a deeper understanding of mobile molecular function.
We have mentioned but not over-asserted "mobile function" in this revision, and discussed how our imaging and mutant analyses indicate the different functions between MEL1 and AGO1b/d in anther development (page 13, lines 7-12).
2"In situ hybridization experiment still needs to be improved, and the specific pollen stage and development stage can not be seen from the cell structure.
As the reviewer requested, we performed in situ hybridization (ISH) for AGO1d localization using a new probe, which has been used in another publication (Fei et al., 2016), and have replaced the images in Figure 6b and Supplementary Fig. 7c and d. The new ISH data enabled us to distinguish the four cell layers of the anther wall, in which the middle layers are thinner than the tapetum and pollen mother cells are less round than those after pachytene. The shape of pollen mother cells and anther wall layers indicate that the stage is between the premeiotic S-phase and the early meiosis.
The staging for pollen needs staining with DAPI or other markers of chromosome formation.
However, since the ISH samples were embedded in paraffin with the whole inflorescence, it is unable to determine the stage. Judging from the structure of the anther wall and pollen mother cells, we have estimated the stage for ISH (Page 11, lines 2-5). The new AGO1d probe information has been added in Supplementary Table 10  There is no relevant evidence to support it. It is recommended to provide the relevant evidence We performed immunopurification (IP) on the guinea pig anti-MEL1 antibody (#1) and, moreover, a mass spectrometry analysis conducted by Kazusa DNA Research Institute confirmed that MEL1 was indeed immunopurified in MEL1-IP fractions, using this guinea pig antibody (Page 9 lines 21-23; Page 19 line 24 -Page 20 line 2). We have also shown the IP-mass spectrometry results in Supplementary Table 9. The guinea pig anti-MEL1 antibody (#1) was used for the immunostaining of Fig. 5 Minor comments# 1" Figure 7C Each cell layer is not marked accordingly. It needs to be corrected We did not realize that the reviewer was referring to Figure 6C instead of Supplementary Fig. 7C for " Figure 7C". Each cell layer in the previous revision ( Supplementary Fig. 7C) was correctly marked.
Therefore, we have added the labels for each cell layer in Figure 6C in this second revision.
3" Supplementary Figure 7. In situ hybridization of AGO1b, AGO1d, MEL1: The pollen development stage in situ hybridization was not indicated. From the figure, a-b and c-d are not in the same stages, and the number of cell layers is different. Each cell layer is not marked accordingly. The in situ hybridization needs to be redone.
We re-performed ISH using a new probe as described above in our response to comment 2. The new ISH data enabled us to distinguish the four cell layers of the anther wall, in which the middle layers are thinner than the tapetum and pollen mother cells are less round than those after pachytene. The shape of pollen mother cells and anther wall layers indicate that the stage is between the premeiotic